Tract Infections In Young Children Research Articles

Age predicts cytokine kinetics and innate immune cell activation following intranasal delivery of IFNγ and GM-CSF in a mouse model of RSV infection

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children and is further associated with increased healthcare utilization and cost of care in the first years of life. Severe RSV disease during infancy has also been linked to the later development of allergic asthma, yet there remains no licensed RSV vaccine or effective treatment. Pre-clinical and clinical studies have shown that disease severity and development of allergic asthma are associated with differences in cytokine production. As a result, stimulation of the innate host immune response with immune potentiators is gaining attention for their prospective application in populations with limited immune responses to antigenic stimuli or against pathogens for which vaccines do not exist. Specifically, macrophage-activating cytokines such as interferon gamma (IFNγ) and granulocyte colony-stimulating factor (GM-CSF) are commercially available immune potentiators used to prevent infections in patients with chronic granulomatous disease and febrile neutropenia, respectively. Moreover, an increasing number of reports describe the protective function of IFNγ and GM-CSF as vaccine adjuvants. Although a positive correlation between cytokine production and age has previously been reported, little is known about age-dependent cytokine metabolism or immune activating responses in infant compared to adult lungs. Here we use a non-compartmental pharmacokinetic model in naïve and RSV-infected infant and adult BALB/c mice to determine the effect of age on IFNγ and GM-CSF elimination and innate cell activation following intranasal delivery.

The Immunotherapeutic Role of Bacterial Lysates in a Mouse Model of Asthma.

Asthma is the most common chronic lower respiratory disease in childhood throughout the world. Recurrent respiratory tract infections in young children, especially viral infections, are the major cause of acute asthmatic exacerbations and contribute to development of asthma. Bacterial extracts have been used to improve the immune defenses of the respiratory tract. However, seldom studies have examined the effect of bacterial lysates on childhood asthma. In this study, we examined whether bacterial lysates (OM-85) will improve symptoms of asthmatic mice via modulation of the immune response. Asthmatic mice models were established with OVA challenge and treated with oral administration of Broncho-Vaxom (OM-85). Next, infiltrations of inflammatory cells including eosinophil and neutrophils were examined. Pulmonary tissues in asthmatic mice models were analyzed by hematoxylin and eosin (HE) staining. The levels of Th1/Th2-typed cytokines in bronchoalveolar lavage fluid (BALF) of asthmatic mice models were examined by enzyme-linked immunosorbent assay. Compared to control group, we found significant reduction of airway wall thickness, luminal stenosis, and mucus plug formation in asthmatic mice models after oral administration of OM-85. The infiltrations of eosinophil were also significantly decreased in BALF in asthmatic mice models. Oral administration of OM-85 was shown to suppress Th2-type cytokine levels. Our findings provide evidence that oral administration of OM-85 is capable of attenuating airway inflammation in asthmatic mice models. Oral administration of OM-85 may have a positive impact in terms of asthma severity.

NF-κB signaling pathway mediates respiratory syncytial virus-induced HMGB1 release in airway epithelial cells to promote an inflammatory response

Abstract Respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in young children, the elderly and immunocompromised individuals. In addition to acute morbidity, RSV infections have been linked to both the development and severity of asthma. No effective treatment or vaccine is currently available for RSV. High mobility group box 1 protein (HMGB1) is a multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. Although HMGB1 is implicated in the pathogenesis of many inflammatory diseases, its involvement in RSV-induced lung inflammation is not yet investigated. The goal of this study is to investigate the molecular mechanisms that induce the release of HMGB1 and the involvement of the NF-κB and TLR4 pathway in RSV pathogenesis. Human lung epithelial cells were infected with RSV in the presence or absence of human recombinant HMGB1 and harvested over time to analyze HMGB1, NF-κB and TLR4 expression by Western blot, immunohistochemistry and qRT-PCR. Our studies with airway epithelial cells clearly show that RSV-induces a significant release of HMGB1 from cells as a result of its translocation from the nucleus to the cytoplasm and subsequently to the extracellular space; that secreted HMGB1 triggers immune responses by activating primary human monocytes via NF-κB-TLR4 signaling pathway. Blockage of one of these molecules could represent a novel therapeutic strategy in the treatment not only for RSV infection, but also for other respiratory diseases that affect human population at any age.

Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.

Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C chemokine motif (182CWAIC186) in the G protein, RSV binds to the corresponding chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log10-fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine.IMPORTANCE RSV binds to the corresponding chemokine receptor, CX3CR1, through a CX3C chemokine motif (182CWAIC186) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A186, within the CX3C motif, mutating it to CX4C (182CWAIAC187), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab')2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.

Parvovirus Expresses a Small Noncoding RNA That Plays an Essential Role in Virus Replication.

Human bocavirus 1 (HBoV1) belongs to the species Primate bocaparvovirus of the genus Bocaparvovirus of the Parvoviridae family. HBoV1 causes acute respiratory tract infections in young children and has a selective tropism for the apical surface of well-differentiated human airway epithelia (HAE). In this study, we identified an additional HBoV1 gene, bocavirus-transcribed small noncoding RNA (BocaSR), within the 3' noncoding region (nucleotides [nt] 5199 to 5338) of the viral genome of positive sense. BocaSR is transcribed by RNA polymerase III (Pol III) from an intragenic promoter at levels similar to that of the capsid protein-coding mRNA and is essential for replication of the viral DNA in both transfected HEK293 and infected HAE cells. Mechanistically, we showed that BocaSR regulates the expression of HBoV1-encoded nonstructural proteins NS1, NS2, NS3, and NP1 but not NS4. BocaSR is similar to the adenovirus-associated type I (VAI) RNA in terms of both nucleotide sequence and secondary structure but differs from it in that its regulation of viral protein expression is independent of RNA-activated protein kinase (PKR) regulation. Notably, BocaSR accumulates in the viral DNA replication centers within the nucleus and likely plays a direct role in replication of the viral DNA. Our findings reveal BocaSR to be a novel viral noncoding RNA that coordinates the expression of viral proteins and regulates replication of viral DNA within the nucleus. Thus, BocaSR may be a target for antiviral therapies for HBoV and may also have utility in the production of recombinant HBoV vectors.IMPORTANCE Human bocavirus 1 (HBoV1) is pathogenic to humans, causing acute respiratory tract infections in young children. In this study, we identified a novel HBoV1 gene that lies in the 3' noncoding region of the viral positive-sense genome and is transcribed by RNA polymerase III into a noncoding RNA of 140 nt. This bocavirus-transcribed small RNA (BocaSR) diverges from both adenovirus-associated (VA) RNAs and Epstein-Barr virus-encoded small RNAs (EBERs) with respect to RNA sequence, representing a third species of this kind of Pol III-dependent viral noncoding RNA and the first noncoding RNA identified in autonomous parvoviruses. Unlike the VA RNAs, BocaSR localizes to the viral DNA replication centers of the nucleus and is essential for expression of viral nonstructural proteins independent of RNA-activated protein kinase R and replication of HBoV1 genomes. The identification of BocaSR and its role in virus DNA replication reveals potential avenues for developing antiviral therapies.

Management of respiratory tract infections in young children\u2014A qualitative study of primary care providers\u2019 perspectives

Respiratory tract infections in young children are the most common cause of general practice visits in Australia. Despite the availability of clinical practice guidelines, the treatment and management of respiratory tract infections in young children is inconsistent. The aim of the study was to explore the management of respiratory tract infections in young children from a multi-disciplinary perspective using across-sectional qualitative research design based on the theoretical domains framework and the Capability, Opportunity and Motivation-B model. In-depth interviews were conducted with 30 primary care providers to explore their knowledge, views and management of respiratory tract infections in young children. Interviews focused on symptomatic management, over-the-counter medications and antibiotic use, and data were thematically analysed. Our findings showed that factors such as primary care providers’ time constraints, parental anxiety, general practitioners’ perception of what parents want, perceived parental pressure, and fear of losing patients were some of the reasons why primary care providers did not always adhere to guideline recommendations. Primary care providers also provided conflicting advice to parents concerning over-the-counter medications and when children should resume normal activities. Overall, this study showed that complex interactions involving emotional and psychological factors influenced the decision making process of primary care providers’ management of respiratory tract infections in young children. A team care approach with consistent advice, and improved communication between primary care providers and parents is vital to overcome some of these barriers and improve guideline adherence. The findings of this research will inform the development of interventions to better manage respiratory tract infections in young children.

The Need for Improved Detection of Urinary Tract Infections in Young Children.

An estimated 400,000 urinary tract infections (UTIs) are diagnosed annually in children aged <3 years in the United States; yet >50% of febrile UTIs may be missed in this population. Here, we explored possible barriers to diagnosing febrile UTIs in very young children through social research of community pediatricians. Following qualitative interviews, a quantitative survey was developed that included a high-risk case for febrile UTI, presented before prompting for the topic of the survey, to gauge practice of delayed testing. Factors associated with delay were explored using univariate logistic regression. The final survey link was sent to three populations via email, with the largest response from a survey sent to pediatricians in Pennsylvania, which formed the basis of our primary results. Of the 218 evaluable responses, 59.6% of physicians would initially test urine in the high-risk case patient, while 21.6% would choose to continue fever reducer and follow-up in 2 days. In the knowledge-based questions, 67.5, 34.0, and 35.6% of respondents identified the correct prevalences in total population, Caucasian girls, and uncircumcised boys, respectively. Many pediatricians (59.5%) believed that delays in detection are common in clinical practice. Physicians who chose to delay testing were more likely to be female, in practice for <25 years, to underestimate prevalence of febrile UTIs and have greater number of children seen per week (all P ≤ 0.02). Our findings support the need for improved communication and education about prevalence in higher risk populations, outcomes associated with delayed diagnosis, and optimal skills for collection of urine in young patients.

Comparison of microbiological diagnosis of urinary tract infection in young children by routine health service laboratories and a research laboratory: Diagnostic cohort study

ObjectivesTo compare the validity of diagnosis of urinary tract infection (UTI) through urine culture between samples processed in routine health service laboratories and those processed in a research laboratory.Population and methodsWe conducted a prospective diagnostic cohort study in 4808 acutely ill children aged <5 years attending UK primary health care. UTI, defined as pure/predominant growth ≥105 CFU/mL of a uropathogen (the reference standard), was diagnosed at routine health service laboratories and a central research laboratory by culture of urine samples. We calculated areas under the receiver-operator curve (AUC) for UTI predicted by pre-specified symptoms, signs and dipstick test results (the “index test”), separately according to whether samples were obtained by clean catch or nappy (diaper) pads.Results251 (5.2%) and 88 (1.8%) children were classified as UTI positive by health service and research laboratories respectively. Agreement between laboratories was moderate (kappa = 0.36; 95% confidence interval [CI] 0.29, 0.43), and better for clean catch (0.54; 0.45, 0.63) than nappy pad samples (0.20; 0.12, 0.28). In clean catch samples, the AUC was lower for health service laboratories (AUC = 0.75; 95% CI 0.69, 0.80) than the research laboratory (0.86; 0.79, 0.92). Values of AUC were lower in nappy pad samples (0.65 [0.61, 0.70] and 0.79 [0.70, 0.88] for health service and research laboratory positivity, respectively) than clean catch samples.ConclusionsThe agreement of microbiological diagnosis of UTI comparing routine health service laboratories with a research laboratory was moderate for clean catch samples and poor for nappy pad samples and reliability is lower for nappy pad than for clean catch samples. Positive results from the research laboratory appear more likely to reflect real UTIs than those from routine health service laboratories, many of which (particularly from nappy pad samples) could be due to contamination. Health service laboratories should consider adopting procedures used in the research laboratory for paediatric urine samples. Primary care clinicians should try to obtain clean catch samples, even in very young children.

Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV.

Molecular Epidemiology of Respiratory Syncytial Virus A G-Protein for 9 Years and Emergence of ON1 Genotype Having 72 Nucleotide Duplication in Korea

BackgroundRespiratory syncytial virus (RSV) is a major pathogen causing seasonal epidemics of lower respiratory tract infection in young children. The attachment (G) glycoprotein is a major virulence factor and a target of human neutralizing antibodies. The G protein shows the evidence of changes of amino acid over time, making its study important in vaccine development strategies. We aimed to explore the molecular epidemiology of G protein of RSV A in Korea.MethodsOne hundred and thirty-six RSV strains were obtained from children who were hospitalized at Seoul National University Children’s Hospital, from October 2005 to September 2014. The frozen stock viruses were propagated in HEp-2 cells and the infected cell lysates were collected when cytopathic effects were apparent. The polymerase chain reaction and sequencing were performed using cDNA constructed from viral RNA and primers from previous studies. Phylogenetic analysis and putative antigenicity plotting were performed using CLC Main Workbench ver. 6.6.5 software (CLC bio, Aarhus, Denmark).ResultsThe entire G-genes were sequenced from all 136 RSV A strains: most (n = 99, 72.8%) strains had 895 nucleotide-span, but the others (n = 45, 27.2%) had 72 nucleotide (nt) duplication, which are well-known ON1 genotype. On phylogenetic grouping, three different genotypes 1–3 except ON1 were additionally defined. During the 2005–2006 season, the prevalence of genotypes 1, 2, and 3 were comparable (36.4%, 27.3%, and 36.4%, respectively), but genotype 1 became predominate (69.2%) in the 2006–2007 season. The genotype 3 increased since 2007–2008 (80.0%) and was identified exclusively during three consecutive seasons thereafter (2009–2011). In the 2011–2012 season, the first ON1 strain was identified in Korea, and the prevalence increased to 91.7% in the 2012–2013 season, then to 100% thereafter (2013–2015). In spite of 72-nt duplication, the putative antigenicity patterns of G protein were comparable between ON1 genotype and the others.ConclusionOur findings highlight the genotype change of circulating RSV A in 3–4 years interval, which might explain the reason for annual local epidemics of RSV A. Also in Korea, RSV A ON1 genotype increased since 2011 and was circulating exclusively since 2013.Disclosures All authors: No reported disclosures.

Molecular detection and characterization of respiratory syncytial virus B genotypes circulating in Pakistani children

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infections in young children, but very little is known about its epidemiology and circulating genotypes in Pakistan. This study analyzed the epidemiological and molecular characteristics of RSV B genotypes in Pakistani children below 5years with acute respiratory tract infections (ARIs) during three consecutive winter seasons from 2010 to 2013. A total of 1941 samples were analyzed for RSV infection by real time PCR and 24% (472/1941) samples were found positive out of which 22.3% (105/472) were sub-typed as RSV-B. The frequency of outpatient cases was higher (62.5%; 295/472) as compared to hospitalized patients (37.5%; 177/472). Patient ages ranged from 2month to 5years with a mean age of 1.48±1.2 (years) and a median age of 1year. Children below one year made up the highest percentage of enrolled subjects and male to female ratio of RSVB positive cases was nearly equivalent (1:1.1). The most common clinical symptoms were cough (96%), fever (80%) and sore throat (50%). All Pak RSVB strains ascribed to the BA genotype showing 91.9–97.1% and 86.2–95.3% homology at the nucleotide and amino acid levels respectively in comparison to BA prototype strain. On phylogenetic analysis, three genotypes of Pakistan RSV B viruses were observed; BA-9 and BA-10 which have been reported previously from other regions, and a third novel genotype assigned as BA-13 which formed a distinct cluster with protein length of 319 AA and showed 9–11 unique AA substitutions. All the RSV B isolates had two potential N-glycosylation sites in HVR2 of G protein and with heavy O-glycosylation of serine and threonine residues (G scores of 0.5–0.7). This study highlights the diversity of RSVB viruses and the significance of RSV as a dominant viral etiologic agent of pediatric ARI. It also emphasizes the need for continued molecular surveillance for early detection of prevalent and newly emerging genotypes to understand epidemiology of RSV infections in various regions of Pakistan.

Use of linked laboratory surveillance and hospital data to estimate the burden of admissions for respiratory syncytial virus infection in children younger than 5 years in England

BackgroundRespiratory syncytial virus (RSV) is a major cause of hospital admission for lower respiratory tract infections in young children. Since virological testing is carried out in only a minority of admitted children, previous estimates of the hospital burden of RSV have relied on time-series modelling techniques. In this study we use a unique linked laboratory surveillance and hospital admission database to estimate the secondary care burden of RSV in children younger than 5 years in England. MethodsNational laboratory surveillance data was probabilistically linked to the Hospital Episode Statistics database for children younger than 5 years in England from Aug 1, 2010, to July 31, 2012, using National Health Service number, date of birth, post code, and sex. We used the linked data to estimate the probability of a respiratory hospital admission being RSV-related based on diagnostic coding and calendar week. Using these probabilities, we estimated the number of RSV-associated admissions by age and sex, and calculated admission rates using Office for National Statistics mid-year population estimates. FindingsWe analysed 4530 RSV-positive linked admissions and 7910 RSV-negative linked admissions. 4257 (94%) of 4530 RSV-positive admissions occurred between Nov 1 and March 31 each year. 3656 (81%) of 4530 RSV-positive admissions were in children younger than 1 year. 3139 (79%) of 4530 RSV-positive admissions had a primary diagnosis of bronchiolitis. Linked admissions had a longer length of stay than did unlinked admissions (median 3 days vs 1). We will present results on the total estimated number of RSV-associated admissions stratified by age, calendar week, and primary diagnosis. Preliminary analysis estimates a total annual average of about 33 500 RSV-associated admissions in children younger than 5 years, with nearly half of these admissions in children younger than 6 months. InterpretationOur study is the first, to our knowledge, to link laboratory and hospital data for RSV in England. Our results point to a likely testing bias towards younger children who are in hospital for longer. Our approach of estimating the national secondary care burden using probability weights from smaller, linked samples could be applied to other seasonal infections. With several RSV vaccines in clinical trials, our results can be used to populate cost-effectiveness models of alternative vaccination strategies. FundingRMR is funded by a Farr Institute of Health Informatics Research PhD studentship.

Respiratory Syncytial Virus Infection Triggers Epithelial HMGB1 Release as a Damage-Associated Molecular Pattern Promoting a Monocytic Inflammatory Response.

RSV is a major cause of serious lower respiratory tract infections in young children and causes severe respiratory morbidity and mortality in the elderly. In addition, to date there is no effective treatment or vaccine available for RSV infection. The mechanisms responsible for RSV-induced acute airway disease and associated long-term consequences remain largely unknown. The oxidative stress response in the airways plays a major role in the pathogenesis of RSV. HMGB1 is a ubiquitous redox-sensitive multifunctional protein that serves as both a DNA regulatory protein and an extracellular cytokine signaling molecule that promotes airway inflammation as a damage-associated molecular pattern. This study investigated the mechanism of action of HMGB1 in RSV infection with the aim of identifying new inflammatory pathways at the molecular level that may be amenable to therapeutic interventions.

Prevailing genotype distribution and characteristics of human respiratory syncytial virus in northeastern China.

Although human respiratory syncytial virus (RSV) is one of the most common viruses inducing respiratory tract infections in young children and the elderly, the genotype distribution and characteristics of RSV in northeastern China have not been investigated. Here, we identified 25 RSV‐A and 8 RSV‐B strains from 80 samples of patients with respiratory infections between February 2015 and May 2015. All 25 RSV‐A viruses were classified as the ON1 genotype, which rapidly spread and became the dominant genotype in the world since being identified in Ontario (Canada) in December 2010. All eight RSV‐B viruses belonged to the BA genotype with a 60‐nucleotide duplication, seven of which formed two new genotypes, BA‐CCA and BA‐CCB. The remaining RSV‐B virus clustered with one of the Hangzhou strains belonging to genotype BA11. Construction of a phylogenetic tree and amino acid substitution analysis showed that Changchun ON1 viruses exclusively constituted Lineages 3, 5 and 6, and contained several unique and newly identified amino acid substitutions, including E224G, R244K, L289I, Y297H, and L298P. Selective pressure was also evaluated, and various N and O‐glycosylation sites were predicted. This study provides the first genetic analysis of RSV in northeastern China and may facilitate a better understanding of the evolution of this virus locally and globally. J. Med. Virol. 89:222–233, 2017. © 2016 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.

Respiratory syncytial virus subtype ON1/NA1/BA9 predominates in hospitalized children with lower respiratory tract infections.

Respiratory syncytial virus (RSV) infection is the leading cause of acute respiratory tract disease in children less than 5 years old. The aim of this study was to further elucidate the molecular properties and clinical characteristics of RSV infection. The study sample included 238 patients <5 years old who were hospitalized with clinical symptoms of upper or lower respiratory tract infection (URTI or LRTI) in the Pediatric Department at the First People's Hospital of Chenzhou, South China in 2014. We subjected nasopharyngeal aspirate (NPA) or nasal swab (NS) samples from the patients to indirect fluorescence assay screens. RSV G genes were amplified by reverse transcription‐PCR (RT‐PCR) and sequenced. Of the 238 patients screened, 64 (26.8%) were confirmed to have RSV infections. Of those 64 confirmed RSV infection cases, 39 (60.9%) had subtype BA9, 13 (20.3%) had the recently identified subtype ON1, 11 (17.2%) had subtype NA1, and 1 (1.6%) had subtype GB2. The predominant presentation was LRTI with coughing, sputum production, fever, and wheezing. RSV subtype NA1 and BA9 infections were found mostly in infants, whereas the age distribution of subtype ON1 infections was more uniform across the age bands. Phylogenetic analysis indicated that, compared with the prototype strain A2, all ON1 and most NA1 isolates had lost one potential N‐glycosylation site at amino acid 251 and 249 due to T251K and N249Y substitution, respectively. These findings suggest that NA1, BA9, and ON1 are the dominant RSV subtypes causing respiratory tract infections in young children presenting to the hospital in South China. J. Med. Virol. 89:213–221, 2017. © 2016 Wiley Periodicals, Inc.

Improving the Diagnosis and Treatment of Urinary Tract Infection in Young Children in Primary Care: Results from the DUTY Prospective Diagnostic Cohort Study.

Up to 50% of urinary tract infections (UTIs) in young children are missed in primary care. Urine culture is essential for diagnosis, but urine collection is often difficult. Our aim was to derive and internally validate a 2-step clinical rule using (1) symptoms and signs to select children for urine collection; and (2) symptoms, signs, and dipstick testing to guide antibiotic treatment. We recruited acutely unwell children aged under 5 years from 233 primary care sites across England and Wales. Index tests were parent-reported symptoms, clinician-reported signs, urine dipstick results, and clinician opinion of UTI likelihood (clinical diagnosis before dipstick and culture). The reference standard was microbiologically confirmed UTI cultured from a clean-catch urine sample. We calculated sensitivity, specificity, and area under the receiver operator characteristic (AUROC) curve of coefficient-based (graded severity) and points-based (dichotomized) symptom/sign logistic regression models, and we then internally validated the AUROC using bootstrapping. Three thousand thirty-six children provided urine samples, and culture results were available for 2,740 (90%). Of these results, 60 (2.2%) were positive: the clinical diagnosis was 46.6% sensitive, with an AUROC of 0.77. Previous UTI, increasing pain/crying on passing urine, increasingly smelly urine, absence of severe cough, increasing clinician impression of severe illness, abdominal tenderness on examination, and normal findings on ear examination were associated with UTI. The validated coefficient- and points-based model AUROCs were 0.87 and 0.86, respectively, increasing to 0.90 and 0.90, respectively, by adding dipstick nitrites, leukocytes, and blood. A clinical rule based on symptoms and signs is superior to clinician diagnosis and performs well for identifying young children for noninvasive urine sampling. Dipstick results add further diagnostic value for empiric antibiotic treatment.

The Diagnosis of Urinary Tract infection in Young children (DUTY): a diagnostic prospective observational study to derive and validate a clinical algorithm for the diagnosis of urinary tract infection in children presenting to primary care with an acute illness.

It is not clear which young children presenting acutely unwell to primary care should be investigated for urinary tract infection (UTI) and whether or not dipstick testing should be used to inform antibiotic treatment. To develop algorithms to accurately identify pre-school children in whom urine should be obtained; assess whether or not dipstick urinalysis provides additional diagnostic information; and model algorithm cost-effectiveness. Multicentre, prospective diagnostic cohort study. Children < 5 years old presenting to primary care with an acute illness and/or new urinary symptoms. One hundred and seven clinical characteristics (index tests) were recorded from the child's past medical history, symptoms, physical examination signs and urine dipstick test. Prior to dipstick results clinician opinion of UTI likelihood ('clinical diagnosis') and urine sampling and treatment intentions ('clinical judgement') were recorded. All index tests were measured blind to the reference standard, defined as a pure or predominant uropathogen cultured at ≥ 10(5) colony-forming units (CFU)/ml in a single research laboratory. Urine was collected by clean catch (preferred) or nappy pad. Index tests were sequentially evaluated in two groups, stratified by urine collection method: parent-reported symptoms with clinician-reported signs, and urine dipstick results. Diagnostic accuracy was quantified using area under receiver operating characteristic curve (AUROC) with 95% confidence interval (CI) and bootstrap-validated AUROC, and compared with the 'clinician diagnosis' AUROC. Decision-analytic models were used to identify optimal urine sampling strategy compared with 'clinical judgement'. A total of 7163 children were recruited, of whom 50% were female and 49% were < 2 years old. Culture results were available for 5017 (70%); 2740 children provided clean-catch samples, 94% of whom were ≥ 2 years old, with 2.2% meeting the UTI definition. Among these, 'clinical diagnosis' correctly identified 46.6% of positive cultures, with 94.7% specificity and an AUROC of 0.77 (95% CI 0.71 to 0.83). Four symptoms, three signs and three dipstick results were independently associated with UTI with an AUROC (95% CI; bootstrap-validated AUROC) of 0.89 (0.85 to 0.95; validated 0.88) for symptoms and signs, increasing to 0.93 (0.90 to 0.97; validated 0.90) with dipstick results. Nappy pad samples were provided from the other 2277 children, of whom 82% were < 2 years old and 1.3% met the UTI definition. 'Clinical diagnosis' correctly identified 13.3% positive cultures, with 98.5% specificity and an AUROC of 0.63 (95% CI 0.53 to 0.72). Four symptoms and two dipstick results were independently associated with UTI, with an AUROC of 0.81 (0.72 to 0.90; validated 0.78) for symptoms, increasing to 0.87 (0.80 to 0.94; validated 0.82) with the dipstick findings. A high specificity threshold for the clean-catch model was more accurate and less costly than, and as effective as, clinical judgement. The additional diagnostic utility of dipstick testing was offset by its costs. The cost-effectiveness of the nappy pad model was not clear-cut. Clinicians should prioritise the use of clean-catch sampling as symptoms and signs can cost-effectively improve the identification of UTI in young children where clean catch is possible. Dipstick testing can improve targeting of antibiotic treatment, but at a higher cost than waiting for a laboratory result. Future research is needed to distinguish pathogens from contaminants, assess the impact of the clean-catch algorithm on patient outcomes, and the cost-effectiveness of presumptive versus dipstick versus laboratory-guided antibiotic treatment. The National Institute for Health Research Health Technology Assessment programme.

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome.

Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases.

Clinical characterisation and phylogeny of respiratory syncytial virus infection in hospitalised children at Red Cross War Memorial Children's Hospital, Cape Town.

BackgroundRespiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children in both the community and hospital setting.MethodsThe clinical presentation, patient and phylogenetic characteristicsof laboratory-confirmed cases of RSV, as well as risk factors for nosocomial infectionat Red Cross War Memorial Children’s Hospital in Cape Town were analysed. A multiplex PCR assay that detects 7 respiratory viruses was used to identify RSV nucleic acid on respiratory specimens.ResultsA total of 226 children were studied, ages ranging between 1 week and 92.5 months (median: 2.8 months, IQR: 1.3–6.3 months) and 51.8 % were males. The median duration of symptoms prior to diagnosis was 2 days (IQR: 1–4 days). Nosocomial infections wereidentified in 22 (9.7 %) children. There were pre-existing medical conditions in 113 (50.0 %) excluding HIV, most commonly prematurity (n = 58, 50.0 %) and congenital heart disease (n = 34, 29.3 %). The commonest presenting symptoms were cough (196, 86.7 %), difficulty in breathing (115, 50.9 %) and fever (91, 41.6 %).A case fatality rate of 0.9 % was recorded. RSV group A predominated (n = 181, 80.1 %) while group B accounted for only 45 (19.9 %) of the infections. The prevalent genotypes were NA1 (n = 127,70.1 %), ON1 (n = 45,24.9 %) and NA2 (n = 9,5.0 %) for group A while the only circulating RSV B genotype was BA4. There was no significant difference in the genotype distribution between the nosocomial and community-acquired RSV infections. Age ≥ 6 months was independently associated with nosocomial infection.ConclusionsA large percentage of children with RSV infection had pre-existing conditions. Approximately one tenth of the infections were nosocomial with age 6 months or older being a risk factor. Though both RSV groups co-circulated during the season, group A was predominant and included the novel ON1 genotype. Continued surveillance is necessary to identify prevalent and newly emerging genotypes ahead of vaccine development and efficacy studies.

Empiric antibiotic treatment for urinary tract infection in preschool children: susceptibilities of urine sample isolates.

Antibiotic treatment recommendations based on susceptibility data from routinely submitted urine samples may be biased because of variation in sampling, laboratory procedures and inclusion of repeat samples, leading to uncertainty about empirical treatment. To describe and compare susceptibilities of Escherichia coli cultured from routinely submitted samples, with E. coli causing urinary tract infection (UTI) from a cohort of systematically sampled, acutely unwell children. Susceptibilities of 1458 E. coli isolates submitted during the course of routine primary care for children <5 years (routine care samples), compared to susceptibilities of 79 E. coli isolates causing UTI from 5107 children <5 years presenting to primary care with an acute illness [systematic sampling: the Diagnosis of Urinary Tract infection in Young children (DUTY) cohort]. The percentage of E. coli sensitive to antibiotics cultured from routinely submitted samples were as follows: amoxicillin 45.1% (95% confidence interval: 42.5-47.7%); co-amoxiclav using the lower systemic break point (BP) 86.6% (84.7-88.3%); cephalexin 95.1% (93.9-96.1%); trimethoprim 74.0% (71.7-76.2%) and nitrofurantoin 98.2% (97.4-98.8%). The percentage of E. coli sensitive to antibiotics cultured from systematically sampled DUTY urines considered to be positive for UTI were as follows: amoxicillin 50.6% (39.8-61.4%); co-amoxiclav using the systemic BP 83.5% (73.9-90.1%); co-amoxiclav using the urinary BP 94.9% (87.7-98.4%); cephalexin 98.7% (93.2-99.8%); trimethoprim 70.9% (60.1-80.0%); nitrofurantoin 100% (95.3-100.0%) and ciprofloxacin 96.2% (89.4-98.7%). Escherichia coli susceptibilities from routine and systematically obtained samples were similar. Most UTIs in preschool children remain susceptible to nitrofurantoin, co-amoxiclav and cephalexin.